It is common knowledge that certain proteins interact with certain other proteins. The interaction between proteins is closely associated with the expression of certain physiological activity and serves as an indicator of the type of physiological activity of a given protein. Thus, when a new protein is discovered, the types of protein with which the new protein interacts provide a substantial hint as to the physiological activity of the protein.
Current well-known methods of analyzing interactions between proteins include immunocoprecipitation, the phage display method, and the yeast two-hybrid system. In immunocoprecipitation, an antibody (anti-A) against a given protein A is prepared and the protein B that coprecipitates with antigen A when anti-A is applied is specified; this method has been widely employed for some time. In the phage display method (for example, Clackson, T., and Wells, J. A., Trends Biotechnol., 12 (1994) 173–184), the phage library, in which various foreign gene fragments are inserted into the coat protein gene of a phage, and expressed as the fused protein with the coat protein, is employed. If a phage expressing protein B is detected using a protein A, the gene for protein B can be specified. In this method, the gene of A can also be specified if the antibody is employed as protein A. Further, when a gene library incorporating random mutations is employed, it is possible to isolate and identify the genes of mutant proteins capable of acting on a specific protein. In the yeast two-hybrid system (Fields, S., et al., Trends Genet. 10 (1994) 286–292), the expression system of a gene library is designed so that when a given protein A acts on another protein B, transcription of the reporter gene employed is activated. In this method, the two functional domains of a DNA binding domain (DB) and a transcription activation domain (TA) are employed to activate transcription, and those proteins activating transcription in the gene expressing fused proteins having both of the domains (for example, when a fused protein of DB and protein A associates with a fused protein of TA and protein B) are screened. Recently, a method of analyzing the interaction between proteins based on surface plasmon resonance employing a laser beam has been developed.
Further, the screening of compounds affecting interaction between proteins, for example, compounds blocking or promoting interaction between proteins, has been conducted with protein pairs for which the interaction between proteins has been clearly specified. In that case, compounds affecting the above-mentioned protein pairs are screened from libraries of compounds comprising a large number of compounds.
The number of genes of human, mouse, and like is to be as high as 60,000 to 100,000. However, the number of genes known to date is still about 10,000, which is low relative to the whole. In recent years, the human genome project has been advancing and the number of genes that are being newly isolated and sequenced is increasing each year. This number is expected to continue to increase in the future. However, the functions of the genes that are being isolated and sequenced are often unknown. Since it is anticipated that all genes will eventually be specified, there is eagerly expected an appearance of the method that can systematically and rapidly analyze the functions of individual genes whose sequences are determined among the huge number of entire genes.
The present inventors conceived of the idea that it is possible to some extent to analyze the functions of genes by examining whether or not proteins having amino acid sequences coded by each gene interact with existing and new proteins.
As set forth above, known methods of analyzing interactions between proteins include immunocoprecipitation, the phage display method, the yeast two-hybrid system, and surface plasmon resonance. However, immunocoprecipitation requires antibodies against individual gene products (proteins). The preparation of antibodies corresponding to the entirety of genes lacks feasibility and there are problems on antibody specificity. In the phage display method and the yeast two-hybrid system, there is a frame restriction on the expression of prescribed genes as fused proteins, and even when the frames match, this does not mean that a specific gene is translated as a complete protein. There are limits to the size of the DNA that can be inserted in phage packaging. And in the yeast two-hybrid system, a DNA transformation into yeast is required and there is considerable inconvenience with regard to the systematic analysis of large numbers of samples. The surface plasmon resonance method requires the purification of each protein and is thus not currently suited to mass production analysis.
Thus, the detection of interaction between large quantities of proteins including new proteins and the selection of proteins that interact is impossible using existing methods.
Further, as set forth above, although there is a known method of screening for compounds affecting protein pairs whose protein—protein interaction has been clearly specified from compound libraries comprising large numbers of compounds, there is no known method of screening pairs of proteins affected by a given compound and the like among pairs of proteins formed as a result of interactions which are thought to be in a large number. Accordingly, it is an object of the present invention to provide a method of readily and rapidly detecting interactions between large numbers of proteins including new proteins.
A further object of the present invention is to provide a method that is capable of simply, efficiently, and systematically detecting which gene products (proteins) interact with each other even when the entire genes have been isolated, that is useful for inferring the functions of proteins.
A still further object of the present invention is to provide a method of screening pairs of proteins that are affected by a given compound from among multiple pairs of proteins.